Estrogens are important for the maintenance of bone mineral density and the prevention of osteoporosis (or low bone mass), a disease that affects more than 29.5 million people in the United States. Estrogen receptor alpha (ER?), in response to estrogens, increases bone mineral density by increasing osteoblast proliferation and differentiation. GATA4, as a pioneer factor (a protein that can bind to target DNA within silent chromatin and initiate chromatin remodeling and helps recruit other transcription factors to the same locus), helps determine these tissue-specific effects of ER? gene regulation in osteoblasts. However, when and where GATA4 binds to DNA, and how GATA4 directs ER? binding to enhancers to regulate osteoblast differentiation is currently unknown. The central hypothesis of this proposal is that GATA4 increases differentiation of mesenchymal stem cells (MSCs) to osteoblasts by opening chromatin for ER? binding to enhancers. This hypothesis will be tested with the following three specific aims. Aim 1 will determine when during differentiation ER? and GATA4 are necessary to regulate osteoblast differentiation using temporally regulated gene expression in vitro and in vivo. Aim 2 will identify where ER? and GATA4 bind to DNA to regulate osteoblast differentiation using state-of-the-art high throughput, whole-genome sequencing technology. Aim 3 will elucidate the mechanism by which GATA4 regulates ER? binding to osteoblast enhancers. Together these experiments will for the first time describe how the novel osteoblast transcription factor GATA4 regulates estrogen biology in bone. These results will have valuable impact through characterization of ER? tissue-specificity in bone and hence will further understanding of how estrogens prevent osteoporosis.